The present invention relates generally to methods for the treatment of immunologically-mediated disorders. In certain embodiments, the invention is related to the use of compositions comprising components prepared from Micobacterium vaccae, Mycobacterium tuberculosis and Mycobacterium smegmatis for the treatment of immunologically-mediated disorders of the respiratory system, such as sarcoidosis, asthma and lung cancers, for treatment of allergic disorders such as atopic dermatitis, for treatment of diseases that benefit from the reduction of eosinophilia, for treatment and prevention of infectious diseases, such as infection with Mycobacterium tuberculosis or Mycobacterium avium, and for the treatment of atherosclerosis, hypercholesterolemia and other disorders that may be improved by modulating IL-10 production.
Tuberculosis is a chronic, infectious disease that is caused by infection with Mycobacterium tuberculosis (M. tuberculosis). It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as a chronic inflammation of the lungs, resulting in fever and respiratory symptoms. If left untreated, significant morbidity and death may result.
Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behavior is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistant mycobacteria.
Inhibiting the spread of tuberculosis requires effective vaccination and accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common mycobacterium employed for this purpose is Bacille Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis (M. bovis). However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public. Diagnosis of M. tuberculosis infection is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, thereby indicating exposure to mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.
A less well-known mycobacterium that has been used for immunotherapy for tuberculosis, and also leprosy, is Microbacterium vaccae (M. vaccae), which is non-pathogenic in humans. However, there is less information on the efficacy of M. vaccae compared with BCG, and it has not been used widely to vaccinate the general public. M. bovis BCG and M. vaccae are believed to contain antigenic compounds that are recognized by the immune system of individuals exposed to infection with M tuberculosis. 
Several patents and other publications disclose treatment of various conditions by administering mycobacteria, including M. vaccae, or certain mycobacterial fractions. U.S. Pat. No. 4,716,038 discloses diagnosis of, vaccination against and treatment of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae. U.S. Pat. No. 4,724,144 discloses an immunotherapeutic agent comprising antigenic material derived from M. vaccae for treatment of mycobacterial diseases, especially tuberculosis and leprosy, and as an adjuvant to chemotherapy. International Patent Publication WO 91/01751 discloses the use of antigenic and/or immunoregulatory material from M. vaccae as an immunoprophylactic to delay and/or prevent the onset of AIDS. International Patent Publication WO 94/06466 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for therapy of HIV infection, with or without AIDS and with or without associated tuberculosis.
U.S. Pat. No. 5,599,545 discloses the use of mycobacteria, especially whole, inactivated M. vaccae, as an adjuvant for administration with antigens that are not endogenous to M. vaccae. This publication theorizes that the beneficial effect as an adjuvant may be due to heat shock protein 65 (hsp65). International Patent Publication WO 92/08484 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of uveitis. International Patent Publication WO 93/16727 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of mental diseases associated with an autoimmune reaction initiated by an infection. International Patent Publication WO 95/26742 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for delaying or preventing the growth or spread of tumors. International Patent Publication WO 91/02542 discloses the use of autoclaved M. vaccae in the treatment of chronic inflammatory disorders in which a patient demonstrates an abnormally high release of IL-6 and/or TNF or in which the patient""s IgG shows an abnormally high proportion of agalactosyl IgG. Among the disorders mentioned in this publication are psoriasis, rheumatoid arthritis, mycobacterial disease, Crohn""s disease, primary biliary cirrhosis, sarcoidosis, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, Guillain-Barre syndrome, primary diabetes mellitus, and some aspects of graft rejection.
M. vaccae is apparently unique among known mycobacterial species in that heat-killed preparations retain vaccine and immunotherapeutic properties. For example, M. bovis BCG vaccines, used for vaccination against tuberculosis, employ live strains. Heat-killed M. bovis BCG and M. tuberculosis have no protective properties when employed in vaccines. A number of compounds have been isolated from a range of mycobacterial species that have adjuvant properties. The effect of such adjuvants is essentially to stimulate a particular immune response mechanism against an antigen from another species.
There are two general classes of compounds that have been isolated from mycobacterial species that exhibit adjuvant properties. The first are water soluble wax D fractions (White et al., Immunology 1:54, 1958; U.S. Pat. No. 4,036,953). The second are muramyl dipeptide-based substances (N-acetyl glucosamine and N-glycolymuramic acid in approximately equimolar amounts) as described in U.S. Pat. Nos. 3,956,481 and 4,036,953. These compounds differ from the delipidated and deglycolipidated M. vaccae (DD-M. vaccae) of the present invention in the following aspects of their composition:
1. They are water-soluble agents, whereas DD-M. vaccae is insoluble in aqueous solutions.
2. They consist of a range of small oligomers of the mycobacterial cell wall unit, either extracted from bacteria by various solvents, or digested from the cell wall by an enzyme. In contrast, DD-M. vaccae comprises processed mycobacterial cells.
3. All protein has been removed from their preparations by digestion with proteolytic enzymes. The only constituents of their preparations are the components of the cell wall peptidoglycan structure, namely alanine, glutamic acid, diaminopimelic acid, N-acetyl glucosamine, and N-glycolylmuramic acid. In contrast, DD-M. vaccae contains 50% w/w protein, comprising a number of distinct protein species.
Sarcoidosis is a disease of unknown cause characterized by granulomatous inflammation affecting many organs of the body and especially the lungs, lymph nodes and liver. Sarcoid granulomata are composed of mononuclear phagocytes, with epithelioid and giant cells in their center, and T lymphocytes. CD4 T lymphocytes are closely associated with the epithelioid cells while both CD4 and CD8 T lymphocytes accumulate at the periphery. The characteristic immunological abnormalities in sarcoidosis include peripheral blood and bronchoalveolar lavage hyper-globulinaemia and depression of xe2x80x98delayed typexe2x80x99 hypersensitivity reactions in the skin to tuberculin and other similar antigens, such as Candida and mumps. Peripheral blood lymphocyte numbers are reduced and CD4:CD8 ratios in peripheral blood are depressed to approximately 1-1.5:1. These are not manifestations of a generalized immune defect, but rather the consequence of heightened immunological activity which is xe2x80x98compartmentalizedxe2x80x99 to sites of disease activity. In patients with pulmonary sarcoidosis, the total number of cells recovered by bronchoalveolar lavage is increased five-to ten-fold and the proportion of lymphocytes increased from the normal of less than 10-14% to between 15% and 50%. More than 90% of the lymphocytes recovered are T lymphocytes and the CD4:CD8 ratio has been reported to be increased from the value of 1.8:1 in normal controls to 10.5:1. The T lymphocytes are predominantly of the Th1 class, producing IFN-xcex3 and IL-2 cytokines, rather than of the Th2 class. Following treatment, the increase in Th1 lymphocytes in sarcoid lungs is corrected.
Sarcoidosis involves the lungs in nearly all cases. Even when lesions are predominantly seen in other organs, subclinical lung involvement is usually present. While some cases of sarcoidosis resolve spontaneously, approximately 50% of patients have at least a mild degree of permanent organ dysfunction. In severe cases, lung fibrosis develops and progresses to pulmonary failure requiring lung transplantation. The mainstay of treatment for sarcoidosis is corticosteroids. Patients initially responding to corticosteroids often relapse and require treatment with other immunosuppressive drugs such as methotrexate or cyclosporine.
Asthma is a common disease, with a high prevalence in the developed world. Asthma is characterized by increased responsiveness of the tracheobronchial tree to a variety of stimuli, the primary physiological disturbance being reversible airflow limitation, which may be spontaneous or drug-related, and the pathological hallmark being inflammation of the airways. Clinically, asthma can be subdivided into extrinsic and intrinsic variants.
Extrinsic asthma has an identifiable precipitant, and can be thought of as being atopic, occupational and drug-induced. Atopic asthma is associated with the enhancement of a Th2-type of immune response with the production of specific immunoglobulin E (IgE), positive skin tests to common aeroallergens and/or atopic symptoms. It can be divided further into seasonal and perennial forms according to the seasonal timing of symptoms. The airflow obstruction in extrinsic asthma is due to nonspecific bronchial hyperesponsiveness caused by inflammation of the airways. This inflammation is mediated by chemicals released by a variety of inflammatory cells including mast cells, eosinophils and lymphocytes. The actions of these mediators result in vascular permeability, mucus secretion and bronchial smooth muscle constriction. In atopic asthma, the immune response producing airway inflammation is brought about by the Th2 class of T cells which secrete IL-4, IL-5 and IL-10. It has been shown that lymphocytes from the lungs of atopic asthmatics produce IL-4 and IL-5 when activated. Both IL-4 and IL-5 are cytokines of the Th2 class and are required for the production of IgE and involvement of eosimophils in asthma. Occupational asthma may be related to the development of IgE to a protein hapten, such as acid anhydrides in plastic workers and plicatic acid in some western red cedar-induced asthma, or to non-IgE related mechanisms, such as that seen in toluene diiusocyanate-induced asthma. Drug-induced asthma can be seen after the administration of aspirin or other non-steroidal anti-inflammatory drugs, most often in a certain subset of patients who may display other features such as nasal polyposis and sinusitis. Intrinsic or cryptogenic asthma is reported to develop after upper respiratory tract infections, but can arise de novo in middle-aged or older people, in whom it is more difficult to treat than extrinsic asthma.
Asthma is ideally prevented by the avoidance of triggering allergens but this is not always possible nor are triggering allergens always easily identified. The medical therapy of asthma is based on the use of corticosteroids and bronchodilator drugs to reduce inflammation and reverse airway obstruction. In chronic asthma, treatment with corticosteroids leads to unacceptable adverse side effects.
Another disorder with a similar immune abnormality to asthma is allergic rhinitis. Allergic rhinitis is a common disorder and is estimated to affect at least 10% of the population. Allergic rhinitis may be seasonal (hay fever) caused by allergy to pollen. Non-seasonal or perennial rhinitis is caused by allergy to antigens such as those from house dust mite or animal dander.
The abnormal immune response in allergic rhinitis is characterized by the excess production of IgE antibodies specific against the allergen. The inflammatory response occurs in the nasal mucosa rather than further down the airways as in asthma. Like asthma, local eosinophilia in the affected tissues is a major feature of allergic rhinitis. As a result of this inflammation, patients develop sneezing, nasal discharge and congestion. In more severe cases, the inflammation extends to the eyes (conjunctivitis), palate and the external ear. While it is not life threatening, allergic rhinitis may be very disabling, prevent normal activities, and interfere with a person""s ability to work. Current treatment involves the use of antihistamines, nasal decongestants and, as for asthma, sodium cromoglycate and corticosteroids.
Atopic dermatitis is a chronic pruritic inflammatory skin disease which usually occurs in families with an hereditary predisposition for various allergic disorders, such as allergic rhinitis and asthma. Atopic dermatitis occurs in approximately 10% of the general population. The main symptoms are dry skin, dermatitis (eczema) localised mainly in the face, neck and on the flexor sides and folds of the extremities accompanied by severe itching. It typically starts within the first two years of life. In about 90% of the patients this skin disease disappears during childhood but the symptoms can continue into adult life. It is one of the commonest forms of dermatitis world-wide. It is generally accepted that in atopy and in atopic dermatitis, a T cell abnormality is primary and that the dysfunction of T cells which normally regulate the production of IgE is responsible for the excessive production of this immunoglobulin.
Allergic contact dermatitis is a common non-infectious inflammatory disorder of the skin. In contact dermatitis, immunological reactions cannot develop until the body has become sensitised to a particular antigen. Subsequent exposure of the skin to the antigen and the recognition of these antigens by T cells result in the release of various cytokines, proliferation and recruitment of T cells and finally in dermatitis (eczema).
Only a small proportion of the T cells in a lesion of allergic contact dermatitis are specific for the relevant antigen. Activated T cells probably migrate to the sites of inflammation regardless of antigen-specificity. Delayed-type hypersensitivity can only be transferred by T cells (CD4+ cells) sharing the MHC class II antigens. The xe2x80x98responsexe2x80x99 to contact allergens can be transferred by T cells sharing either MHC class I (CD8+ cells) or class II (CD4+ cells) molecules (Sunday, M. E. et al., J. Immunol. 125:1601-1605, 1980). Keratinocytes can produce interleukin-1 which can facilitate the antigen presentation to T cells. The expression of the surface antigen intercellular adhesion molecule-1 (ICAM-1) is induced both on keratinocytes and endothelium by the cytokines tumor necrosis factor (TNF) and interferon-gamma (IFN-xcex3).
If the causes can be identified, removal alone will cure allergic contact dermatitis. During active inflammation, topical corticosteroids are useful. An inhibitory effect of cyclosporin has been observed in delayed-type hypersensitivity on the pro-inflammatory function(s) of primed T cells in vitro (Shidani, B. et al., Eur. J. Immunol. 14:314-318, 1984). The inhibitory effect of cyclosporin on the early phase of T cell activation in mice has also been reported (Milon, G. et al., Ann. Immunol. (Inst. Pasteur) 135d:237-245, 1984).
Lung cancer is the leading cause of death from cancer. The incidence of lung cancer continues to rise and the World Health Organization estimates that by 2000AD there will be 2 million new cases annually. Lung cancers may be broadly classified into two categories: small cell lung cancer (SCLC) which represents 20-25% of all lung cancers, and non-small cell lung cancer (NSCLC) which accounts for the remaining 75%. The majority of SCLC is caused by tobacco smoke. SCLC tends to spread early and 90% of patients present at diagnosis with involvement of the mediastinal lymph nodes in the chest. SCLC is treated by chemotherapy, or a combination of chemotherapy and radiotherapy. Complete response rates vary from 10% to 50%. For the rare patient without lymph node involvement, surgery followed by chemotherapy may result in cure rates exceeding 60%. The prognosis for NSCLC is more dismal, as most patients have advanced disease by the time of diagnosis. Surgical removal of the tumor is possible in a very small number of patients and the five year survival rate for NSCLC is only 5-10%.
The factors leading to the development of lung cancer are complex and multiple. Environmental and genetic factors interact and cause sequential and incremental abnormalities that lead to uncontrolled cell proliferation, invasion of adjacent tissues and spread to distant sites. Both cell-mediated and humoral immunity have been shown to be impaired in patients with lung cancer. Radiotherapy and chemotherapy further impair the immune function of patients. Attempts have been made to immunize patients with inactivated tumor cells or tumor antigens to enhance host anti-tumor response. Bacille Calmette-Guerin (BCG) has been administered into the chest cavity following lung cancer surgery to augment non-specific immunity. Attempts have been made to enhance anti-tumor immunity by giving patients lymphocytes treated ex vivo with interleukin-2 (IL-2). These lymphokine-activated lymphocytes acquire the ability to kill tumor cells. Current immunotherapies for lung cancer are still at a developmental stage and their efficacies have yet to be established for the standard management of lung cancer.
Atherosclerosis is a chronic inflammatory disease of the arterial wall that is characterized by accumulation of lipids, macrophages, T lymphocytes, smooth muscle cells and extracellular matrix. Anti-inflammatory cytokines are produced during the inflammatory reaction and are believed to modulate the inflammatory process. Interleukin-10 (IL-10) is secreted by Th2 lymphocytes and by macrophages, and is known to have anti-inflammatory properties. Mallat et al. recently reported studies in which IL-10 was shown to have a protective effect in the formation and stability of atherosclerotic lesions in mice (Circ. Res. 1999, 85:el7-24). These studies suggest that agents that increase IL-10 production may be employed to modulate the extent and/or severity of atherosclerosis.
Other disorders in which administration of IL-10 has been shown to beneficial include hypercholesterolemia (see U.S. Pat. No. 5,945,097); bacterial infections, including infection with gram-negative and/or gram-positive bacteria (see U.S. Pat. Nos. 5,837,293 and 5,837,232); and insulin-dependent diabetes mellitus (see, U.S. Pat. No. 5,827,513). In addition, U.S. Pat. No. 5,871,725 discloses a method of treating cancer by administering to a patient peripheral blood mononuclear cells (PBMC) that have been activated with IL-10.
Briefly stated, the present invention provides compositions and methods for the prevention and treatment of immunologically-mediated disorders, including disorders of the respiratory system (such as infection with mycobacteria such as M. tuberculosis or M. avium, sarcoidosis, asthma, allergic rhinitis and lung cancers), allergic disorders such as atopic dermatitis, diseases that benefit from the reduction of eosinophilia, and disorders that may be improved by modulating IL-10 production (such as atherosclerosis, hypercholesterolemia, cancer, bacterial infections and insulin-dependent diabetes mellitus).
In a first aspect, compositions comprising delipidated and deglycolipidated mycobacterial cells are provided. In specific embodiments, the delipidated and deglycolipidated cells are prepared from M. vaccae, M. tuberculosis and/or M. smegmatis. 
In a second aspect, the present invention provides compositions comprising a derivative of delipidated and deglycolipidated mycobacterial cells, the derivative of delipidated and deglycolipidated mycobacterial cells being selected from the group consisting of: delipidated and deglycolipidated mycobacterial cells that have been treated by alkaline hydrolysis; delipidated and deglycolipidated mycobacterial cells that have been treated by acid hydrolysis; and delipidated and deglycolipidated mycobacterial cells that have been treated with periodic acid. In preferred embodiments, such compositions comprise a derivative of delipidated and deglycolipidated M. vaccae cells, the derivative of delipidated and deglycolipidated M. vaccae cells being selected from the group consisting of: delipidated and deglycolipidated M. vaccae cells that have been treated by alkaline hydrolysis; delipidated and deglycolipidated M. vaccae cells that have been treated by acid hydrolysis; and delipidated and deglycolipidated M. vaccae cells that have been treated with periodic acid. The derivatives of delipidated and deglycolipidated M. vaccae preferably contain galactose in an amount less than 9.7% of total carbohydrate, more preferably less than 5% of total carbohydrate, and most preferably less than 3.5% total carbohydrate. In certain embodiments, the derivatives of delipidated and deglycolipidated M. vaccae contain glucosamine in an amount greater than 3.7% of total carbohydrate, preferably greater than 5% total carbohydrate and more preferably greater than 7.5% total carbohydrate.
In further aspects of this invention, methods are provided for the treatment of a disorder in a patient, including disorders of the respiratory system, such methods comprising administering to the patient a composition of the present invention. In certain embodiments, the disorder is selected from the group consisting of mycobacterial infections, asthma, sarcoidosis, allergic rhinitis and lung cancers. In one embodiment, the compositions are administered to the airways leading to or located within the lungs, preferably by inhalation through the nose or mouth, and are preferably administered in aerosol forms. The compositions may also, or alternatively, be administered by intradermal, transdermal or subcutaneous routes.
In another aspect, the present invention provides methods for the treatment of a disorder of the respiratory system in a patient by the administration of a composition of the present invention, wherein the disorder is characterized by the presence of eosinophilia in the tissues of the respiratory system. Examples of such diseases include asthma and allergic rhinitis. In a related aspect, the present invention provides methods for the reduction of eosinophilia in a patient, such methods comprising administering at least one of the compositions disclosed herein. Typically, the reduction in eosinophilia will vary between about 20% and about 80%. The percentage of reduction in lung eosinophilia can be determined by measuring the number of eosinophils in bronchoalveolar lavage fluid before and after treatment as described below.
In yet a further aspect, methods for enhancing the production of IL-10 are provided, such methods comprising administering a composition of the present invention. As discussed above, it has recently been shown that IL-10 plays a protective role in the formation and stability of atherosclerotic lesions. IL-10 has also been shown to be effective in the treatment of hypercholesterolemia, cancer, bacterial infections, and insulin-dependent diabetes mellitus. The inventive compositions may thus be usefully employed in the treatment of such disorders.
The present invention further provides methods for the activation of xcex3xcex4 T cells, xcex3xcex4 T cells or NK cells, and thereby repairing epithelium, in a patient, comprising administering to the patient a composition of the present invention.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.